During synaptic transmission, neurotransmitters stored in presynaptic vesicles are released by excocytosis through fusion of vesicles with the plasma membrane. In a subsequent step, membranes and proteins at the synapse are reinternalized by a reverse process, endocytosis. In the analysis of this synaptic vesicle cycle genetically encoded pH-sensitive fluorescent proteins like the GFP-derivative pHluorin have become indispensable tools. These probes are capable of detecting changes in pH that accompany excocytosis and subsequent reacidification of endocytosed vesicles. Here we describe a new class of fluorescent probes, based on pH-sensitive organic dyes coupled to phospholipids, as promising alternative to genetically encoded fluorescent proteins like pHluorin. Moreover the pH-dependent fluorescence properties of these dyes are opposite to those of pHluorin. In hippocampal neurons, cell membranes can be stained in a pH dependent manner, and upon quenching of the fluorescence at the plasma membrane by a slightly basic pH, vesicle recycling can be monitored yielding fluorescence transients with kinetics mirroring those of the well characterized pHluorin signal. Furthermore, this approach can be used to study vesicle recycling in acute preparations like bipolar cells of the retina, where application of genetically encoded probes was not possible so far. This experimental approach using pH-dependent fluorescent lipids has not only the potential of being used in a variety of cellular and slice preparations, but in addition will shed light on an important presynaptic mechanism neglected so far, namely lipid recycling. Comparison of vesicle incorporation of different dye-labeled lipid moieties will bring new insights into lipid organisation and trafficking at the synapse.